Increasing soil temperature to reduce sclerotial viability of
Sclerotium cepivorum
in New Zealand soils
K.L. McLean*, J. Swaminathan, A. Stewart
Soil, Plant and Ecological Sciences Division, Plant Sciences Group, P.O.Box 84, Lincoln University, Canterbury, New Zealand.
Received 2 November 1999; received in revised form 13 March 2000; accepted 7 June 2000
Abstract
A preliminary laboratory-based trial indicatedSclerotium cepivorumsclerotial viability could be reduced from.96±10.7% after 28 d at 208C and to 0% after 16 d at 308C. Soil solarisation signi®cantly reducedS.cepivorumsclerotial viability in two separate trials in Canterbury (Wakanui silt loam soil), New Zealand (to 40.2 and 53.3%, respectively) when soil was covered with clear 50mm thick polythene for
4 weeks. Sclerotial viability further decreased in two New Zealand sites; Canterbury (to 8.7%) and Blenheim (shallow silt loam soil) (to 0%) when the soil was solarised for an 8 week period. Solarisation increased the soil temperature by 6±78C in Canterbury, although the highest temperatures were recorded in Blenheim. Microorganisms isolated from the recovered sclerotia included species ofTrichoderma, Verti-cillium,Fusarium,Mucor,Aspergillusand four unidenti®ed bacterial species.q2001 Elsevier Science Ltd. All rights reserved.
Keywords:Sclerotium cepivorum; Sclerotia; Solarisation; Soil temperature; New Zealand
1. Introduction
Onion white rot is the most destructive disease of
Allium-species worldwide and is caused by the soilborne pathogen
Sclerotium cepivorumBerk. In the past, soil applications of
dicarboximide and triazole systemic fungicides have given adequate control of onion white rot but, in recent years, their effectiveness has declined due to enhanced microbial degra-dation of the chemicals (Slade et al., 1992). The build up of
S. cepivorumsclerotia in the soil may also contribute to the
decline in fungicide effectiveness. A control measure such as soil solarisation could be used to relieve disease pressure by decreasing the number of viable sclerotia in the soil. Soil solarisation could be integrated into a disease management programme for effective control of onion white rot.
Soil solarisation was pioneered in Israel (Katan et al., 1976) and California (Pullman et al., 1979). The technique involves levelling the soil with minimal soil compaction before thorough wetting, which increases the thermal sensi-tivity of the soil micro¯ora and fauna as well as increasing heat transfer or conduction in the soil (Mahrer et al., 1984). The soil is then covered with thin clear polyethylene
sheet-ing dursheet-ing the hottest months of the year. Increases in soil temperature can then eliminate or at least reduce soilborne pathogen inoculum as well as insects, nematodes and weed
seeds. Clear polythene (50mm thick) is most commonly
used as coloured polythene tends to absorb heat rather than allow it to be transmitted into the soil (DeVay, 1991). Successful reductions in S. cepivorum sclerotial viability have been reported from Egypt (Satour et al., 1989; 1991), Spain (Basallote-Ureba and Melero-Vara, 1993) and Austra-lia (Porter and Merriman, 1985). New Zealand has a climate that is marginal for soil solarisation compared to many parts of the world where it is practiced. A preliminary laboratory-based soil temperature trial indicated the range of
tempera-tures, which were lethal to S. cepivorum sclerotia. These
results were in agreement with results obtained by Adams (1987) in the United States of America. When these tempera-tures were compared with soil temperatempera-tures from a soil solar-isation weed control trial (Alexander, 1990) in Canterbury, the results indicated that soil solarisation may be a viable control option for onion white rot in New Zealand.
The use of soil solarisation to control onion white rot is novel in New Zealand. This paper reports ®rst on the results of a preliminary laboratory-based soil temperature trial examining the in¯uence of constant soil temperatures on sclerotial viability. Then, results are given of three soil solarisation trials undertaken in Canterbury (1995, 1996,
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1997) and one trial in Blenheim (1997), which are two of New Zealand's main onion and garlic growing regions.
2. Methods and materials
2.1. Production of inoculum
Sclerotia ofS. cepivorum(E68-isolated from an infected onion, Pukekohe, Auckland, NZ in 1990 (trial 1) and SC 3-isolated from an infected onion, Pukekohe, Auckland, NZ in November 1996 (trials 2, 3 and 4)) were produced on whole wheat grains (Alexander and Stewart, 1994) and harvested after 8 weeks using progressive wet sieving through 850 and 500mm sieves. Only sclerotia retained on the 500mm sieve
were used in the trials and the sclerotia were air dried on sterile Whatman no. 1 ®lter paper for 24 h. A sample of 100 sclerotia were surface sterilised in 0.25% NaOCl for 1 min, washed in three changes of sterile distilled water (SDW), touched to Whatman no. 1 ®lter paper using sterile forceps to absorb excess liquid and placed onto potato dextrose agar (PDA) droplets in Petri dishes. The sclerotia were incubated at 208C in the dark and examined daily for 10 d to determine viability. Sclerotial viability for all trials was recorded as .96%.
2.2. The in¯uence of constant soil temperatures on sclerotial viability
Sclerotia were counted into lots of 100. Each lot was placed in a polyester mesh bag (Scarpa Filtration Ltd.,
Auckland, New Zealand; 85mm pore size, 10£10 cm2
with a marker attached for location purposes.
Plastic containers 18£18£19 cm3 were ®lled to
within 1 cm of the top with coarsely sieved, air-dried,
unsterile Wakanui silt loam soil (bulk density:
1.01 g cm23, McLaren and Cameron, 1996). The soil was
wetted to 25% and maintained throughout the course of the trial to simulate ®eld conditions. Three bags of sclerotia were buried in each plastic container at a depth of 10 cm. Although the edges of each bag overlapped, the sclerotia were positioned in each bag to ensure they were sandwiched between soil. Seven plastic containers were maintained at each of the following temperatures: 20, 30, 35, 40, 45 and 508C, a range of temperatures similar to those used by other researchers (Porter and Merriman, 1983; Adams, 1987). A reference treatment was included where sclerotia were maintained at 208C in a glass vial in the dark for the duration of the trial. For each temperature, three bags of sclerotia were randomly selected at each of seven assessment times: 6, 12 h, 1, 2, 8, 16 and 28 d.
The sclerotia were recovered from the bags and surface sterilised in 0.25% NaOCl for 1 min, washed in ®ve changes of SDW and blotted dry on sterile Whatman no. 1 ®lter paper. The sclerotia were then placed individually using sterile forceps onto isolated PDA droplets in Petri dishes. The dishes were sealed with polythene wrap and incubated
in the dark at 208C. Sclerotial germination was recorded every second day for 10 d. The percentage viability of scler-otia, relative to the number buried, was determined. Results were analysed using an analysis of variance (ANOVA) with temperature and length of exposure period as variables. The appropriateness of an ANOVA for this data was checked by visual inspection of a residual plot. This plot adequately con®rmed the normality and homogeneity of the variance of the data. A Fishers Least Signi®cant Difference test was used for pairwise comparisons. Probit analysis was performed to determine the exposure period required at each temperature to reduce sclerotial viability by 95%
(LD95). The data were converted to thermal days
(temperature£length of exposure period in days) to
compare the effects of short exposure periods at high temperatures with longer exposure periods at lower temperatures on sclerotial viability.
2.3. Soil solarisation trial design
For each of the four trials conducted, sclerotia were counted into lots of 50. Each lot was placed in a polyester mesh bag as previously described. A reference treatment where sclerotia were maintained in a glass vial at 208C in the dark for the duration of the trials was included for each trial.
Trials 1 and 2 each ran for four week periods over two consecutive summers (Trial 1: 13/12/95±10/1/96, Trial 2: 11/1/97±11/2/97) and were conducted at a ®eld site at Lincoln University, Canterbury, New Zealand in Wakanui silt loam soil. In trial 1, each of the eight plots 3£6 m2
contained six bags of sclerotia buried equidistant from each other. Three of the bags were randomly selected and buried at a depth of 10 cm and the remaining three bags were buried at 20 cm. In trial 2, each of the eight plots 2£ 3 m2 contained three bags of sclerotia buried equidistant from each other at a depth of 10 cm.
Trials 3 and 4 ran for two 8 week periods (10/12/97±2/2/ 98 and 15/12/97±12/2/98, respectively). Trial 3 was conducted at a ®eld site at Lincoln University, Canterbury, New Zealand and trial 4 was conducted at the Marlborough Research Centre, Blenheim, New Zealand in shallow silt loam soil. Each of the eight plots 3£3 m2 contained three bags of sclerotia buried equidistant from each other at a depth of 10 cm. The polyester mesh bags contained 20 g sieved Wakanui silt loam soil (#200mm particle size) and
20 g quartz sand in addition to the 50 sclerotia.
Following burial of the sclerotia, all plots were irrigated to saturation and on the following day, 50mm thick
trans-parent polythene (Permathane Plastics, Auckland, New Zealand) (Satour et al., 1989) was laid over four randomly selected plots. The edges of the polythene were buried in the soil to a depth of 10 cm. The remaining four uncovered plots were sprinkler irrigated once a week and weeds were removed by hand.
For trial 1, temperature sensors (Philips KTY83-110) encased in stainless steel tubes were placed in the soil to a
depth of 10 and 20 cm in each of the eight plots. The soil temperature was recorded every 30 min using a ®eld data logger (Datataker DT 600) for the duration of the trial. For trials 2, 3 and 4 a Tiny Tag temperature logger with internal sensor (Gemini Dataloggers, Chichester, UK) was enclosed in a plastic container, wrapped in a plastic bag and buried 10 cm deep in one solarised and one non-solarised plot. Temperatures were recorded every 30 min (trial 2) and every 3 h (trials 3 and 4).
Sclerotia were retrieved from the bags at trial completion and assessed for viability as previously described. Sclerotial germination was recorded every second day and percentage viability of sclerotia, relative to the number buried, was determined. Any other microorganisms growing out into the agar droplets were also noted and identi®ed where possi-ble. For trial 1, the results were analysed using an ANOVA with solarised and non-solarised treatments and depth of burial as variables. For trials 2, 3 and 4 the results were analysed using an ANOVA with solarised and non-solarised treatments as variables.
3. Results
3.1. The in¯uence of constant soil temperatures on sclerotial viability
There was a signi®cant reduction P,0:05in sclerotial
viability when the sclerotia were incubated at different temperatures and also after varying lengths of exposure periods (Table 1). There was also a signi®cant interaction between the soil temperatures and the length of exposure period. There was no signi®cant reduction P,0:05 in
sclerotial viability in the reference treatment for the duration of the trial. At 208C, sclerotial viability was signi®cantly reduced P,0:05after 28 d incubation with only 10.7%
of the sclerotia remaining viable. At 308C, sclerotial viabi-lity was reduced P,0:05to 0% after incubation for 16 d.
At 358C, 8 d incubation was required to reduce sclerotial viability to 0%. Incubation at 408C for 6 h was suf®cient to reduce P,0:05sclerotial viability to 31% with a
reduc-tion to 0% after only 1 d. At 458C, a 12 h incubation period reduced P,0:05sclerotial viability to zero and at 508C,
sclerotial viability was lost within the ®rst 6 h incubation period.
Short exposure periods at higher temperatures reduced sclerotial viability by 95% (LD95) (Table 2). In contrast,
longer exposure periods of 16±28 d were required to reduce sclerotial viability by 95% at lower temperatures (24.8 and 24.38C, respectively).
When the data were modelled by probit analysis, equiva-lent thermal day periods did not result in equivaequiva-lent losses in sclerotial viability. For example, after exposure to 358C for 8 d, sclerotial viability was reduced to 21% but only to 52% after exposure to 208C for 14 d, for the same thermal day period (280). Similarly, after 2 d at 408C (80 thermal days), only 3% of the sclerotia remained viable, whereas 79% of the sclerotia remained viable after 4 d at 208C.
3.2. Soil solarisation trials
The mean daily maximum temperatures for solarised and non-solarised soil for trials 1, 2 and 3 are shown in Figs. 1± 3. There is no soil temperature pro®le for trial 4 as the Tiny Tag temperature logger in the solarised plot malfunctioned during the course of the trial and the temperature data could not be retrieved. The maximum and mean soil temperatures at 10 cm in the solarised soil ranged from 39.2±42.78C and
K.L. McLean et al. / Soil Biology & Biochemistry 33 (2001) 137±143
Table 1
The mean percentage of viableSclerotium cepivorumsclerotia recovered from soil at a range of temperatures after selected exposure periods in the laboratory
n300
Temperature (8C) Days
0.25 0.5 1 2 8 16 28
a Mean values followed by the same letter do not differ signi®cantly P
,0:05within each column and across each row according to a Fishers Least Signi®cant Difference test, LSD16.64.
Table 2
Temperature required at selected exposure periods to reduceSclerotium cepivorumsclerotial viability by 95% in laboratory soil (modelled by probit analysis)
Temperature (8C) Exposure period (days) (LD95)
24.6±28.88C, respectively. In the non-solarised soil, the maximum and mean soil temperatures at 10 cm ranged
from 27±33.68C and 18.9±23.38C, respectively. Maximum
soil temperatures at 10 cm were higher P,0:05 in the
solarised soil than the non-solarised soil for trials 1 and 2 but not for trial 3. The maximum soil temperature at 20 cm for the solarised and non-solarised soil were also signi®cantly different P,0:05 for trial 1. For trial 1, the maximum
temperature at 10 and 20 cm in the solarised plots differed by a maximum of 48C on any one day (Fig. 1). In the non-solarised plots, the maximum temperatures differed by a
maximum of 18C on any one day. Soil temperatures of
208C and greater were maintained for 1897, 484 and
777 h in solarised soil compared with 930.5, 279 and 648 h in non-solarised soil for trials 1, 2 and 3, respectively (Table 3).
K.L. McLean et al. / Soil Biology & Biochemistry 33 (2001) 137±143
Fig. 1. The mean daily maximum soil temperature for solarised and non-solarised soil at 10 and 20 cm, Canterbury (trial 1).
The results for percentage sclerotial recovery and viabi-lity for all four trials are presented in Table 4. For trial 1, the depth at which the sclerotia were buried did not signi®cantly affect P,0:05sclerotial recovery or viability, therefore,
the number of sclerotia recovered at 10 and 20 cm were combined for analysis.
The percentage of recovered sclerotia was signi®cantly less (P,0.05) from the solarised plots than from the refer-ence treatment for all trials except trial 1. In trial 1 there was no signi®cant difference (P,0.05) in sclerotial recovery between the reference, non-solarised control and solarised treatments. In trial 4, in addition to fewer P,0:05
scler-otia being recovered from the solarised plots, fewer P,
0:05 sclerotia were recovered from the non-solarised
control plots compared with the reference treatment. Fragments of sclerotial rind remained in the bags and vials, which indicated that the unrecovered sclerotia had disintegrated.
Sclerotial viability was reduced signi®cantly P,0:05
in the solarised plots in all four trials (Table 4). In trial 1 and 4, there was also a signi®cant reduction P,0:05 in
sclerotial viability between the non-solarised control and reference treatments.
A number of fungal species emerged from the sclerotia in all trials when plated onto agar droplets. The fungi were identi®ed as isolates ofVerticillium,Penicillium,Fusarium,
Trichoderma,Aspergillus,Mucorand an isolate of
Paecilo-myces lilacinus (Thom) Samson. Unidenti®ed bacterial
species were also present. Both the solarised and non-solarised sclerotia were colonised by the microorganisms in all trials.
4. Discussion
Increases in constant soil temperature in the laboratory caused a decrease in sclerotial viability. Sclerotia readily decayed and lost viability when incubated for short periods
K.L. McLean et al. / Soil Biology & Biochemistry 33 (2001) 137±143
Fig. 3. The mean daily maximum soil temperature for solarised and non-solarised soil, Canterbury (trial 3).
Table 3
Total number of hours recorded above selected temperatures and the maximum and mean temperature in solarised and non-solarised soil for four ®eld soil solarisation trials at 10 cm depth (ND Ð no data)
Temperature (8C) Trial 1-Canterburya Trial 2-Canterbury Trial 3-Canterbury Trial 4 -Blenheim
Solarised Non-solarised Solarised Non-solarised Solarised Non-solarised Solarised Non-solarised
$40 13 0 0 0 51 0 ND 0
$35 140 0 63 0 105 9 ND 0
$30 551 0 99 1 240 21 ND 54
$20 1897 931 484 279 777 648 ND 993
Maximum temperature 42.7 27 39.2 27.9 41.3 33.6 ND 33
Mean temperature 27.6 20.3 24.6 18.9 28.8 21.1 ND 23.3
at temperatures above 408C. Adams (1987) reported a simi-lar result with continuous soil temperatures of 508C for 4 h and 458C for 12 h, lethal to S. cepivorum sclerotia. The equivalent thermal day data indicated that although higher temperatures maintained for short periods of time may have the same thermal days as lower temperatures maintained for longer periods, the resulting number of viable sclerotia was not equivalent. This has important implications in that higher temperatures are required for a rapid decrease in sclerotial viability. Temperatures greater than 408C were recorded in Canterbury and it is probable that soil
tempera-tures reached 408C in Blenheim. However, the signi®cant
reduction inS. cepivorumsclerotial recovery and viability is more likely due to the effect of ¯uctuating sub-lethal temperatures rather than high temperatures.
The detrimental effects of ¯uctuating sub-lethal tempera-tures have been well documented (Katan et al., 1976; Pull-man et al., 1979; Porter and MerriPull-man, 1983). It is most probable that both thermal in¯uence and biological control activity reduced sclerotial viability in these soil solarisation trials. Fluctuating temperatures may increase sclerotial vulnerability to soil microorganisms or increase heat resis-tant saprophyte populations subsequently increasing the parasitic and lytic effects on the sclerotia (Katan et al., 1976). The increased microbial activity could explain why pieces of sclerotial rind remained in many of the bags.
Increased colonisation of S. cepivorum sclerotia by soil
microorganisms, following treatment with sub-lethal
temperatures has also been reported (Entwistle and Muna-singhe, 1990). The presence of soil microorganisms on the recovered sclerotia may suggest that the sclerotia were in a weakened state.Aspergillusis reported as a possible second-ary colonist of sclerotia (Phillips, 1990) and is tolerant of high soil temperatures (Dwivedi, 1991). Bacterial species have also been reported to colonise cracks in the sclerotial rind of weakened sclerotia (Lifshitz et al., 1983).
With New Zealand's climate, the effects of ¯uctuating sub-lethal temperatures are more in¯uential in reducing sclerotial viability than high temperatures. An 8 week solar-isation treatment was more effective in reducing sclerotial viability than a 4 week period as the sclerotia were subjected to sub-lethal temperatures for twice as long. The lower viability from the 8 week trials might also have been
related to the addition of soil and sand to the bags. The polyester mesh bags may have provided an insulating effect in trials 1 and 2 that was overcome in trials 3 and 4. If an insulating effect had occurred then it is likely that the loss of sclerotial viability in natural populations would be higher than the reported data.
Soil solarisation reduced sclerotial viability by 91.3 and 100% in trial 3 and 4, respectively. All sclerotia were at a depth of 10 cm. While sclerotia are able to infect
Allium-species when placed up to 30 cm deep in the soil, it is the sclerotia in the top 10 cm of soil that mainly contribute to disease spread (Crowe and Hall, 1980). Reducing sclerotial viability in the top 10 cm of the soil would therefore ease disease pressure inAlliumcrops. In addition, soil tempera-tures at 10 and 20 cm were almost identical and solarising a
S. cepivorum naturally infested ®eld would still subject
sclerotia at soil depths greater than 20 cm to sub-lethal temperature ¯uctuations. While the effects would not be as great deeper in the soil, the sclerotia may still be weakened and more vulnerable to invasion by antagonistic microorganisms.
Within New Zealand, the use of soil solarisation to control onion white rot will be most suited to the garlic growing regions in Blenheim where soil temperatures and sunshine hours are generally higher than elsewhere in New Zealand. Trials are now required to determine the actual reduction in onion white rot afforded by this technique in the ®eld. The combination of soil solarisation with biologi-cal control agents may provide more effective control of onion white rot than the use of soil solarisation alone.
Acknowledgements
This research was supported by the Brian Mason Science and Technical Trust.
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Table 4
PercentageSclerotium cepivorumsclerotial recovery and viability from all treatments within each of the four ®eld soil solarisation trials. Percentage values followed by the same letter do not differ signi®cantly P,0:05within columns (n300 (trial 1),n150 (trials 2, 3 and 4))
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% Recovery % Viabilityb % Recovery % Viability % Recovery % Viability % Recovery % Viability
Reference 100 a 100 a 100 a 98.8 a 90.6 a 88.6 a 96.0 a 89.3 a
Non-solarised control 96.5 a 66.3 b 94.2 a 88.3 a 74.7 a 70.6 a 61.7 b 49.7 b
Solarised 88.3 a 40.2 c 59.0 b 53.3 b 46.0 b 8.7 b 29.0 c 0 c
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